Neutrophils play a critical role in development of LPS-induced airway disease

We investigated the role of neutrophils in the development of endotoxin-induced airway disease via systemic neutrophil depletion of C3H/HeBFeJ mice and coincident inhalation challenge with lipopolysaccharide (LPS) over a 4-wk period. Mice were made neutropenic with intraperitoneal injections of neutrophil antiserum before and throughout the exposure period. Experimental conditions included LPS-exposed, antiserum-treated; LPS-exposed, control serum-treated; air-exposed, antiserum-treated; and air-exposed, control serum-treated groups. Physiological, biological, and morphological assessments were performed after a 4-wk exposure and again after a 4-wk recovery period. After the 4-wk exposure, LPS-induced inflammation of the lower airways was significantly attenuated in the neutropenic mice, although airway responsiveness (AR) to methacholine (MCh) remained unchanged. After the recovery period, LPS-exposed neutrophil-replete mice had increased AR to MCh when compared with the LPS-exposed neutropenic animals. Morphometric data indicate that the 4-wk exposure to LPS leads to a substantial expansion of the subepithelial area of the medium-sized airways (90-129 microm diameter) in nonneutropenic mice but not neutropenic mice, and this difference persisted even after the recovery period. Expression of bronchial epithelial and subepithelial transforming growth factor-beta1 (TGF-beta1) was diminished in the challenged neutropenic mice compared with the neutrophil-sufficient mice. These studies demonstrate that neutrophils play a critical role in the development of chronic LPS-induced airway disease.     

IL-10 reduces grain dust-induced airway inflammation and airway hyperreactivity.

To determine whether interleukin-10 (IL-10) could alter the development of grain dust-induced airway disease, we pretreated mice with either saline or IL-10 intravenously, exposed the mice to an inhalation challenge with corn dust extract (CDE), and measured inflammation and the development of airway hyperreactivity. Pretreatment with IL-10, in comparison to saline, reduced the concentration and percentage of polymorphonuclear cells in the lavage fluid 30 min after the inhalation challenge with CDE (P < 0. 05). In comparison to saline-treated mice, IL-10 did not significantly alter the degree of airway hyperreactivity 30 min after the exposure to CDE. IL-10-treated mice lavaged 18 h after challenge with CDE also exhibited a lower percentage of polymorphonuclear cells in the lavage fluid (P < 0.05) and had significantly less airway hyperreactivity than did mice pretreated with the saline placebo (P < 0.05). These findings indicate that exogenous IL-10 is effective in reducing airway inflammation and airway hyperreactivity due to the inhalation of CDE.     

Subchronic endotoxin inhalation causes persistent airway disease

The endotoxin component of organic dusts causes acute reversible airflow obstruction and airway inflammation. To test the hypothesis that endotoxin alone causes airway remodeling, we have compared the response of two inbred mouse strains to subchronic endotoxin exposure. Physiological and biological parameters were evaluated after 1 day, 5 days, or 8 wk of exposure to endotoxin [lipopolysaccharide (LPS)] in endotoxin-sensitive (C3HeB/FeJ) and endotoxin-resistant (C3H/HeJ) mice. After 5 days or 8 wk of LPS exposure, only C3HeB/FeJ had elevated airway hyperreactivity to inhaled methacholine. Only the C3HeB/FeJ mice had significant inflammation of the lower respiratory tract after 1 day, 5 days, or 8 wk of LPS exposure. Stereological measurements of small, medium, and large airways indicated that an 8-wk exposure to LPS resulted in expansion of the submucosal area only in the C3HeB/FeJ mice. Cell proliferation as measured by bromodeoxyuridine incorporation contributed to the expansion of the submucosa and was only significantly elevated in C3HeB/FeJ mice actively exposed to LPS. C3HeB/FeJ mice had significantly elevated levels of interleukin-1beta protein in whole lung lavage after 1 day and 5 days of LPS exposure and significantly elevated protein levels of total and active transforming growth factor-beta1 in whole lung lavage fluid after 5 days of LPS exposure. Our findings demonstrate that subchronic inhalation of LPS results in the development of persistent airway disease in endotoxin-responsive mice.     

Toll-like receptor 4 antagonist (E5564) prevents the chronic airway response to inhaled lipopolysaccharide

Although chronic inhalation of endotoxin or lipopolysaccharide (LPS) causes all of the classic features of asthma, including airway hyperreactivity, airway inflammation, and airway remodeling, the mechanisms involved in this process are not clearly understood. The objective of this study was to determine whether intratracheal treatment with LPS antagonist (E5564, a lipid A analog) prevented the development of chronic endotoxin-induced airway disease in a mouse model of environmental airway disease. Pretreatment with 10 and 100 microg of E5564 was found to inhibit the airway response (hyperreactivity and inflammation) for up to 48 h after the administration of the compound. Repeated dosing with 50 microg of E5564 intratracheally did not cause any measurable toxicity. Therefore, in a chronic experiment, mice were treated with either E5564 (50 microg) or vehicle three times weekly for 5 wk and simultaneously daily exposed to either LPS (4.65 +/- 0.30 microg/m3) or saline aerosol. E5564 was effective in decreasing the airway hyperreactivity to methacholine, the air space neutrophilia, the interleukin-6 in the lung lavage fluid, and the neutrophil infiltration of the airways 36 h after 5 wk of LPS inhalation. Less collagen deposition was observed in the airways of E5564-treated mice compared with vehicle-treated mice after a 4-wk recovery period. Our results indicate that E5564, a Toll-like receptor 4 antagonist, minimizes the physiological and biological effects of chronic LPS inhalation, suggesting a therapeutic role for competitive LPS antagonists in preventing or reducing endotoxin-induced environmental airway disease.     

Bronchial hyperreactivity is associated with enhanced grain dust-induced airflow obstruction.

Bronchial hyperreactivity (BHR) is associated with the presence of airway inflammation in asthma and is seen in individuals occupationally exposed to grain dust. To better understand the relationship between BHR and pulmonary inflammation after grain dust exposure, we carried out an inhalation challenge to corn dust extract (CDE) on seven subjects with BHR [a 20% or greater decrease in forced expiratory volume in 1 s (FEV(1)) compared with diluent FEV(1) with a cumulative dose of histamine 相似文献   

Fibrinolysis in LPS-induced chronic airway disease

To examine the role of the fibrinolytic system in LPS-induced airway disease, we compared the effect of a chronic LPS challenge in plasminogen activator inhibitor-deficient (C57BL/6JPAI-1-/-) mice and wild-type (WT) C57BL/6J mice. Physiological and biological assessments were performed, immediately after, and 4 wk after an 8-wk exposure to LPS or saline. Immediately after the LPS exposure, WT mice had increased estimates of airway reactivity to methacholine compared with C57BL/6JPAI-1-/- mice; however, airway inflammation was similar in both LPS-exposed groups. Significant increases in both active transforming growth factor (TGF)-beta1 and active matrix metalloproteinase (MMP)-9 was detected after LPS exposure in WT but not C57BL/6JPAI-1-/- mice. C57BL/6JPAI-1-/- mice showed significantly less TGF-beta1 in the lavage and higher MMP-9 in the lung tissue than WT mice at the end of exposure and 4 wk later. After LPS exposure, both WT and C57BL/6JPAI-1-/- mice had substantial expansion of the subepithelial area of the medium [diameter (d) = 90-129 microm]- and large (d > 129 microm)-size airways when compared with saline-exposed mice. Subepithelial fibrin deposition was prevalent in WT mice but diminished in C57BL/6JPAI-1-/-. PAI-1 expression by nonciliated bronchial epithelial cells was enhanced in LPS-exposed WT mice compared with the saline-exposed group. Four weeks after LPS inhalation, airway hyperreactivity and the expansion of the subepithelial area in the medium and large airways persisted in WT but not C57BL/6JPAI-1-/- mice. We conclude that an active fibrinolytic system can substantially alter the development and resolution of the postinflammatory airway remodeling observed after chronic LPS inhalation.     

A high docosahexaenoic acid diet alters lung inflammation and recovery following repetitive exposure to aqueous organic dust extracts

Agricultural workers, especially those who work in swine confinement facilities, are at increased risk for developing pulmonary diseases including asthma, chronic obstructive pulmonary disease, and chronic bronchitis due to exposures to fumes, vapors, and organic dust. Repetitive exposure to agricultural dust leads to unresolved inflammation, a common underlying mechanism that worsens lung disease. Besides occupational exposure to dusts, diet also significantly contributes to inflammation and disease progression. Since DHA (docosahexaenoic acid), a polyunsaturated omega-3 fatty acid and its bioactive metabolites have key roles in inflammation resolution, we rationalized that individuals chronically exposed to organic dusts can benefit from dietary modifications. Here, we evaluated the role of DHA in modifying airway inflammation in a murine model of repetitive exposure to an aqueous extract of agricultural dust (three-week exposure to swine confinement dust extract, HDE) and after a one-week resolution/recovery period. We found that mice fed a high DHA diet had significantly increased bronchoalveolar lavage fluid (BALF) levels of DHA-derived resolvins and lower TNFα along with altered plasma levels of endocannabinoids and related lipid mediators. Following the one-week recovery we identified significantly reduced BALF cellularity and cytokine/chemokine release along with increased BALF amphiregulin and resolvins in DHA diet-fed versus control diet-fed mice challenged with HDE. We further report observations on the effects of repetitive HDE exposure on lung Ym1+ and Arg-1+ macrophages. Overall, our findings support a protective role for DHA and identify DHA-derived resolvins and endocannabinoids among the potential mediators of DHA in altering airway inflammation in chronic agricultural dust exposure.     

Combined budesonide/formoterol therapy in conjunction with allergen avoidance ameliorates house dust mite-induced airway remodeling and dysfunction

Allergic asthma is characterized by airway inflammation in response to chronic allergen exposure, resulting in remodeling of the airway wall accompanied by dysfunctional airway physiology. However, a link between the immune-inflammatory response to allergen and changes to airway structure and physiology has not yet been fully elucidated. Moreover, the impact of inhaled corticosteroids and beta(2)-agonists, the primary pharmacotherapy for asthma, on this process has not been completely evaluated. In this study, we employed a murine model of chronic exposure to a common environmental aeroallergen, house dust mite, to recapitulate the phenotype of clinical asthma. By examining the therapeutic effects of corticosteroid/beta(2)-agonist combination therapy with budesonide/formoterol (BUD/FORM) in this model of airway disease, we endeavored to determine the impact of BUD/FORM on lung inflammation, structure, and physiology. BUD/FORM was delivered either while allergen exposure was ongoing (concurrent therapy) or following the cessation of allergen exposure (postexposure therapy). Our results show that airway inflammation was substantially reduced in BUD/FORM-treated mice in the concurrent therapy group, whereas in the postexposure therapy group airway inflammation spontaneously resolved. In contrast, BUD/FORM was most effective in resolving several aspects of airway remodeling and bronchial hyperreactivity when delivered in conjunction with allergen withdrawal. This study demonstrates that although both BUD/FORM therapy and allergen avoidance independently reduce airway inflammation, only BUD/FORM therapy in conjunction with allergen avoidance can effectively reverse airway remodeling and bronchial hyperreactivity induced by chronic allergen exposure.     

Eotaxin represents the principal eosinophil chemoattractant in a novel murine asthma model induced by house dust containing cockroach allergens

Asthma represents a serious health problem particularly for inner city children, and recent studies have identified that cockroach allergens trigger many of these asthmatic attacks. This study tested the concept that asthma-like pulmonary inflammation may be induced by house dust containing cockroach allergens. An aqueous extract was prepared from a house dust sample containing endotoxin and high levels of cockroach allergens. BALB/c mice were immunized with the house dust extract (HDE) and received two additional pulmonary challenges. Bronchoalveolar lavage (BAL) eosinophil counts and eotaxin levels were significantly increased in immunized mice exposed to the HDE, whereas neutrophils were the predominant BAL inflammatory cell in the unimmunized mice. Kinetics studies in immunized mice demonstrated a peak pulmonary inflammatory response 48 h after the last challenge. The allergic response in this model was further confirmed by histological and physiological studies demonstrating a significant influx of eosinophils and lymphocytes in the peribronchial area, and severe airway hyperreactivity through whole-body plethysmography. The specificity of the response was established by immunizing with HDE and challenging with purified cockroach allergen, which induced pulmonary eosinophilia and airway hyperreactivity. Ab inhibition of eotaxin significantly inhibited the number of BAL eosinophils. These data describe a novel murine model of asthma-like pulmonary inflammation induced by house dust containing endotoxin and cockroach allergens and further demonstrate that eotaxin represents the principal chemoattractant for the recruitment of the pulmonary eosinophils.     

Allergen-induced airway disease is mouse strain dependent

We investigated the development of airway hyperreactivity (AHR) and inflammation in the lungs of nine genetically diverse inbred strains of mice [129/SvIm, A/J, BALB/cJ, BTBR+(T)/tf/tf, CAST/Ei, C3H/HeJ, C57BL/6J, DBA/2J, and FVB/NJ] after sensitization and challenge with ovalbumin (OVA). At 24, 48, and 72 h post-OVA exposure, the severity of AHR and eosinophilic inflammation of the mouse strains ranged from relatively unresponsive to responsive. The severity of the airway eosinophilia of some strains did not clearly correlate with the development of AHR. The temporal presence of T helper type 2 cytokines in lung lavage fluid also varied markedly among the strains. The levels of IL-4 and IL-13 were generally increased in the strains with the highest airway eosinophilia at 24 and 72 h postexposure, respectively; the levels of IL-5 were significantly increased in most of the strains with airway inflammation over the 72-h time period. The differences of physiological and biological responses among the inbred mouse strains after OVA sensitization and challenge support the hypothesis that genetic factors contribute, in part, to the development of allergen-induced airway disease.     

Endotoxemia and hepatic injury in a rodent model of hindlimb unloading.

Hindlimb unloading (HU) is known to induce physiological alterations in various organ systems that mimic some responses observed after exposure to microgravity. In the present study, the effects of up to 4 wk of HU on the liver were assessed in male Wistar rats and two mouse strains: endotoxin-sensitive C57BL/6 mice and endotoxin-resistant C3H/HEJ mice. Plasma levels of endotoxin, a known stimulator of hepatic injury, were measured in portal and systemic blood samples. Endotoxin was elevated by approximately 50% in portal blood samples of mice and rats but was not detectable in systemic blood. This low-grade portal endotoxemia was associated with hepatic injury in rats and C57BL/6 mice as indicated by inflammation and elevated serum transaminase activities. Blood levels of the cytokine TNF-alpha were increased by approximately 50% in C57BL/6 mice; no significant elevation of this cytokine was detected in rats. Messenger RNA levels of the acute-phase proteins serum amyloid A, haptoglobin, and lipopolysaccharide binding protein were significantly enhanced after 3 wk of HU in endotoxin-sensitive rodents. In contrast, no histological changes or significant increases in serum enzyme activity were detected after HU in C3H/HEJ mice despite portal endotoxin levels of 222 +/- 83.4 pg/ml. At the 3-wk time point, expression of acute-phase proteins was not elevated in C3H/HEJ mice; however, expression after 4 wk of HU was similar to endotoxin-sensitive rodents. In conclusion, these findings indicate that HU induced mild portal endotoxemia, which contributed to the observed hepatic injury in endotoxin-sensitive rodents.     

Tolerance induced by chronic inhaled antigen in a murine asthma model is not mediated by endotoxin

Ovalbumin (OVA)-sensitized wildtype (WT) and endotoxin-resistant (ER) mice developed similar degrees of airways eosinophilia and serum OVA-specific IgE levels after acute aerosolized OVA challenge. WT mice demonstrated methacholine hyperreactivity, whereas ER mice showed no change in responsiveness. With chronic aerosolized OVA challenge, both WT and ER mice developed local tolerance, with resolution of airway eosinophilia but persistence of anti-OVA IgE in serum. Thus, the development of local tolerance with chronic aerosol exposure to OVA is independent of any potential effects of endotoxin in the OVA aerosol solution.     

Endotoxin Exposure during Sensitization to Blomia tropicalis Allergens Shifts TH2 Immunity Towards a TH17-Mediated Airway Neutrophilic Inflammation: Role of TLR4 and TLR2

Experimental evidence and epidemiological studies indicate that exposure to endotoxin lipopolysaccharide (eLPS) or other TLR agonists prevent asthma. We have previously shown in the OVA-model of asthma that eLPS administration during alum-based allergen sensitization blocked the development of lung TH2 immune responses via MyD88 pathway and IL-12/IFN-γ axis. In the present work we determined the effect of eLPS exposure during sensitization to a natural airborne allergen extract derived from the house dust mite Blomia tropicalis (Bt). Mice were subcutaneously sensitized with Bt allergens co-adsorbed onto alum with or without eLPS and challenged twice intranasally with Bt. Cellular and molecular parameters of allergic lung inflammation were evaluated 24 h after the last Bt challenge. Exposure to eLPS but not to ultrapure LPS (upLPS) preparation during sensitization to Bt allergens decreased the influx of eosinophils and increased the influx of neutrophils to the airways. Inhibition of airway eosinophilia was not observed in IFN-γdeficient mice while airway neutrophilia was not observed in IL-17RA-deficient mice as well in mice lacking MyD88, CD14, TLR4 and, surprisingly, TLR2 molecules. Notably, exposure to a synthetic TLR2 agonist (PamCSK4) also induced airway neutrophilia that was dependent on TLR2 and TLR4 molecules. In the OVA model, exposure to eLPS or PamCSK4 suppressed OVA-induced airway inflammation. Our results suggest that B. tropicalis allergens engage TLR4 that potentiates TLR2 signaling. This dual TLR activation during sensitization results in airway neutrophilic inflammation associated with increased frequency of lung TH17 cells. Our work highlight the complex interplay between bacterial products, house dust mite allergens and TLR signaling in the induction of different phenotypes of airway inflammation.     

Antifungal and airway remodeling roles for murine monocyte chemoattractant protein-1/CCL2 during pulmonary exposure to Asperigillus fumigatus conidia

Asperigillus fumigatus spores or conidia are quickly eliminated from the airways of nonsensitized individuals but persist in individuals with allergic pulmonary responsiveness to fungus. A. fumigatus-induced allergic airway disease is characterized by persistent airway hyperreactivity, inflammation, and fibrosis. The present study explored the role of CCR2 ligands in the murine airway response to A. fumigatus conidia. Nonsensitized and A. fumigatus-sensitized CBA/J mice received an intratracheal challenge of A. fumigatus conidia, and pulmonary changes were analyzed at various times after conidia. Whole lung levels of monocyte chemoattractant protein-1 (MCP-1/CCL2), but neither MCP-3/CCL7 nor MCP-5/CCL12, were significantly elevated at days 3 and 7 after conidia in nonsensitized mice. MCP-1/CCL2 was significantly increased in lung samples from A. fumigatus-sensitized mice at days 14 and 30 after a conidia challenge. Administration of anti-MCP-1/CCL2 antiserum to nonsensitized mice for14 days after the conidia challenge attenuated the clearance of conidia and significantly increased airway hyperreactivity, eosinophilia, and peribronchial fibrosis compared with nonsensitized mice that received conidia and normal serum. Adenovirus-directed overexpression of MCP-1/CCL2 in A. fumigatus-sensitized mice markedly reduced the number of conidia, airway inflammation, and airway hyperresponsiveness at day 7 after the conidia challenge in these mice. Immunoneutralization of MCP-1/CCL2 levels in A. fumigatus-sensitized mice during days14-30 after the conidia challenge did not affect the conidia burden but significantly reduced airway hyperreactivity, lung IL-4 levels, and lymphocyte recruitment into the airways compared with the control group. These data suggest that MCP-1/CCL2 participates in the pulmonary antifungal and allergic responses to A. fumigatus conidia.     

Chronic tobacco smoke exposure increases airway sensitivity to capsaicin in awake guinea pigs.

Tobacco smoke (TS) exposure induces airway hyperreactivity, particularly in sensitive individuals with asthma. However, the mechanism of this airway hyperreactivity is not well understood. To investigate the relative susceptibility of atopic and nonatopic individuals to TS-induced airway hyperreactivity, we exposed ovalbumin (OA)-sensitized and nonsensitized guinea pigs to TS exposure (5 mg/l air, 30-min exposure, 7 days/wk for 120-156 days). Two similar groups exposed to compressed air served as controls. Airway reactivity was assessed as an increase in enhanced pause (Penh) units using a plethysmograph that allowed free movement of the animals. After 90 days of exposure, airway reactivity increased in OA-TS guinea pigs challenged with capsaicin, bradykinin, and neurokinin A fragment 4--10 aerosols. In addition, substance P content increased in lung perfusate of OA-TS guinea pigs in response to acute TS challenge compared with that of the other groups. Airway hyperirritability was not enhanced by phosphoramidon but was attenuated by a cocktail of neurokinin antagonists, nor was airway hyperreactivity observed after either methacholine or histamine challenge in OA-TS guinea pigs. Chronic TS exposure enhanced neither airway reactivity to histamine or methacholine nor contractility of isolated tracheal rings. In conclusion, chronic TS exposure increased airway reactivity to capsaicin and bradykinin aerosol challenge, and OA-TS guinea pigs were most susceptible to airway dysfunction as the result of exposure to TS compared with the other groups. Increased airway reactivity to capsaicin suggests a mechanism involving neurogenic inflammation, such as increased activation of lung C fibers.     

Human apolipoprotein E genotypes differentially modify house dust mite-induced airway disease in mice

Apolipoprotein E (apoE) is an endogenous negative regulator of airway hyperreactivity (AHR) and mucous cell metaplasia in experimental models of house dust mite (HDM)-induced airway disease. The gene encoding human apoE is polymorphic, with three common alleles (ε2, ε3, and ε4) reflecting single amino acid substitutions at amino acids 112 and 158. The objective of this study was to assess whether the human apoE alleles modify airway responses to repeated nasal HDM challenges. Mice expressing the human apoE ε2 (huApoE2), ε3 (huApoE3), or ε4 (huApoE4) alleles received nasal HDM challenges, and airway responses were compared with mice expressing the endogenous murine apoE gene (muApoE). huApoE3 mice displayed significant reductions in AHR, mucous cell metaplasia, and airway inflammation compared with muApoE mice. The attenuated severity of airway inflammation in huApoE3 mice was associated with reductions in lung mRNA levels of Th2 and Th17 cytokines, as well as chemokines (CCL7, CCL11, CCL24). huApoE4 mice had an intermediate phenotype, with attenuated AHR and IgE production, compared with muApoE mice, whereas airway inflammation and mucous cell metaplasia were not reduced. In contrast, HDM-induced airway responses were not modified in mice expressing the huApoE2 allele. We conclude that the polymorphic huApoE alleles differentially modulate HDM-induced airway disease, which can be stratified, in rank order of increasing disease severity, ε3 < ε4 < ε2. These results raise the possibility that the polymorphic apoE alleles may modify disease severity in human asthma.     

Divergent immune responses to house dust mite lead to distinct structural-functional phenotypes

Asthma is a chronic airway inflammatory disease that encompasses three cardinal processes: T helper (Th) cell type 2 (Th2)-polarized inflammation, bronchial hyperreactivity, and airway wall remodeling. However, the link between the immune-inflammatory phenotype and the structural-functional phenotype remains to be fully defined. The objective of these studies was to evaluate the relationship between the immunologic nature of chronic airway inflammation and the development of abnormal airway structure and function in a mouse model of chronic asthma. Using IL-4-competent and IL-4-deficient mice, we created divergent immune-inflammatory responses to chronic aeroallergen challenge. Immune-inflammatory, structural, and physiological parameters of chronic allergic airway disease were evaluated in both strains of mice. Although both strains developed airway inflammation, the profiles of the immune-inflammatory responses were markedly different: IL-4-competent mice elicited a Th2-polarized response and IL-4-deficient mice developed a Th1-polarized response. Importantly, this chronic Th1-polarized immune response was not associated with airway remodeling or bronchial hyperresponsiveness. Transient reconstitution of IL-4 in IL-4-deficient mice via an airway gene transfer approach led to partial Th2 repolarization and increased bronchial hyperresponsiveness, along with full reconstitution of airway remodeling. These data show that distinct structural-functional phenotypes associated with chronic airway inflammation are strictly dependent on the nature of the immune-inflammatory response.     

Expression of airway hyperreactivity to acetylcholine as a simple autosomal recessive trait in mice

An increased airway response to various bronchoconstricting agents is one of the hallmarks of asthma. An interdependence of heredity and environment appears to determine this nonspecific hyperreactivity of the airways. The present study describes the patterns of inheritance of the airway response to a direct mediator of smooth muscle contraction (acetylcholine) in A/J and C3H/HeJ inbred mice and their offspring. The mean airway response to acetylcholine was greater than sixfold higher in A/J mice as compared with C3H/HeJ mice. Two phenotypes were easily distinguished on the basis of airway responses to acetylcholine in the progeny of A/J and C3H/HeJ mice. These two phenotypes were termed HYPERREACTIVE (after the A/J strain) and HYPOREACTIVE (after the C3H/HeJ strain). The observed frequencies of HYPERREACTIVE and HYPOREACTIVE phenotypes in the (A/J x C3H/HeJ) F1; (C3H/HeJ x A/J) F1 x C3H/HeJ (C3H/HeJ backcross); and the [(A/J x C3H/HeJ) F1 x (C3H/HeJ x A/J) F1] F2 are consistent with a single autosomal recessive gene primarily controlling acetylcholine-mediated airway responses. This single gene difference in airway response is completely inhibited by atropine and therefore mediated entirely by the muscarinic acetylcholine receptor.